|Authors: ||K.E. Keller, N.J. Mosier, A.L. Thomas, D.F. Quito-Avila, R.R. Martin|
|Keywords: ||next generation sequencing (NGS), Illumina, Sambucus, American hop latent virus, Helleborous mosaic virus, Poplar mosaic virus|
Cuttings of nine elderberry (Sambucus spp.) cultivars were sent from Missouri (USA) to the USDA-ARS laboratory in Corvallis, Oregon (USA) to be tested for the presence of viruses.
Double-stranded RNA (dsRNA) was extracted from the nine cultivars and all showed a similar electrophoretic banding pattern, with bands of about 8000 base pairs (bp) in addition to several smaller bands.
The dsRNA from ‘Bob Gordon’ was used as template for next generation sequencing (NGS) and further analysis.
DsRNA was converted to cDNA using reverse transcription with degenerate oligonucleotide primers.
The cDNA was further amplified by PCR, prepared for NGS using the Illumina format.
Sequencing yielded approximately 75 million 80 bp, paired end reads.
Reads were assembled into contigs using SCRAPE.pl and two large contigs of 8521 and 8425 nucleotides were obtained.
Each contig displayed significant levels of identity to several carlaviruses and about 50% nucleotide sequence identity between each other.
From nucleotide sequence and translated amino acid sequence data, it is clear that there are two distinct carlaviruses infecting elderberry.
Elderberry carlavirus 145 (EBCV145) showed less homology to known carlaviruses both at the nucleotide and amino acid level.
Elderberry carlavirus 153 (EBCV153) had numerous coding regions that were homologous to American hop latent virus. Specific primers were developed for each of the two carlaviruses.
All nine cultivars tested were positive for both viruses with the exception of ‘Marge’, which only had EBCV145. Meristem tips were collected from ‘Wyldewood’ and used to regenerate plants in tissue culture.
Plants were obtained that tested negative for one or both carlaviruses, demonstrating the ability to produce elderberries free of these viruses.
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